The endoplasmic reticulum (ER) is a continuous membrane network of flat sheets and curved tubules. The network plays a central role in a variety of processes critical for human health, ranging from lipid and protein synthesis and transport, to calcium homeostasis. Segregation of certain ER processes into rough and smooth sub-domains as well as the striking differences in the morphology and organization of ER membranes in specialized cell types are suggestive of a link between organelle form and function. Yet a clear understanding of the mechanisms generating and maintaining the sub-compartmentalized tubular and cisternal membrane network of the ER is lacking. Our long-term objective is to define the molecular underpinnings and functional roles for the distinct structural features of the mammalian ER. The first step in achieving this goal is identification of the proteins that govern ER morphology. Although a number of candidates likely to participate in ER structuring have been identified, few have been shown to be required in living cells. To facilitate the search for the required proteins, a candidate-based morphological RNAi screen in cultured cells has been initiated. A recently published early secretory pathway proteome of 1430 constituents (Gilchrist et al, 2006, Cell 127:1265-1281) has been pared down to a candidate pool of 117 proteins, the majority of which are previously uncharacterized. The screen is proving effective, as it has already yielded two hits and preliminary analysis of the first is confirming its role in regulating ER structure. The primary objective of this exploratory proposal is to continue the screen to identify new ER structure regulators. Each hit to emerge from the screen will be confirmed by testing whether expression of an RNAi-immune construct coding for the targeted protein rescues the RNAi-induced ER morphological abnormalities. Once hits are validated, site-directed mutagenesis, protein interaction assays, and phenotypic analysis aided by high-resolution imaging will enable identification of the structural determinants and binding partners required for the ability of each newly identified protein to perform its organelle structuring function. Preliminary studies on the first confirmed hit to emerge from the screen have identified both a critical amino acid residue in the ER structure regulator and a likely binding partner required to mediate its function. The completion of the proposed screen and preliminary analysis of the proteins identified under this exploratory award promises to yield novel insights into the mechanisms that generate ER network architecture. Ultimately, an understanding of the functional consequences of the RNAi-induced structural deficits or rearrangements identified through the screen may lead to novel therapeutics in the treatment of diseases that stem from ER dysfunction. PUBLIC HEALTH RELEVANCE: The endoplasmic reticulum (ER) carries out a myriad of processes critical to human health and disease. It is the site of drug detoxification;lipid and protein biosynthesis and trafficking;protein folding and quality control;and calcium signaling. The functional importance of the organelle in all tissues is clear. Moreover the striking differences in the architecture and organization of the ER membrane network in specialized cells types have led to the long-standing notion that the structure and sub-compartmentalization of the organelle are critical for its functions. Yet the mechanisms that structure the ER and the exact relationship between organelle structure and function are only poorly understood. The objective of this proposal is to identify novel mammalian proteins that govern ER structure. The results are expected to lead to a fuller understanding of the structure-function relationship of the organelle. Ultimately, it is hoped that the insights gained will aid in the development of therapeutics for the treatment of diseases that stem from ER dysfunction.